Field of the Invention
The present invention relates to the use of polypeptides having α-xylosidase activity and belonging to the glycoside hydrolase family 31 (GH31, www.cazy.org & PMID: 18838391) Cantarel et al.), especially the GH31 α-xylosidase designated BACOVA 03422.
Nucleic Acids Res. 2009 January; 37 (Database issue):D233-8. doi: 10.1093/nar/gkn663. Epub 2008 Oct. 5.
The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics.
Cantarel B L, Coutinho P M, Rancurel C, Bernard T, Lombard V, Henrissat B., and polynucleotides encoding the polypeptides and catalytic domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides and catalytic domains.
Description of the Related Art
α-xylosidases of which the sequence is known belong to family GH31. To date, three bacterial GH31 α-xylosidases have been characterized (CjXyl31A, Larsbrink et al. 2011, Biochem. J. 436:567-580; YicI, Okuyama et al. 2004, Protein Expr. Purif. 37:170-179; XylQ, Chaillou et al. 1998, J. Bacteriol. 180:2312-2320). In archea, one α-xylosidase has been described (XylS, Moracci et al. 2000, J. Biol. Chem. 275:22082-22089). Two fungal GH31 α-xylosidases have also been described, Aspergillus niger (AxIA, Scott-Craig et al. 2011, J. Biol. Chem. 286:42848-42854) and an Aspergillus nidulans α-xylosidase (Bauer et al. 2006, Proc Natl. Acad. Sci. U.S.A. 130:11417-11422). The A. niger AxIA has been shown to enhance the yield of fermentable sugars on some substrates when supplemented to commercial cellulase preparations. The mechanism of action was assumed to be in aiding in xyloglucan hydrolysis (Jabbour et al. 2013, Biotechnol. Biofuels 6:online version).
In plants, several GH31 α-xylosidase have been described (AXyl1 and Brassica GH31, Sampedro et al. 2001, Plant Physiol. 126:910-920 and Iglesias et al. 2006, Plant Cell Physiol. 47:55-63; from Tropaeolum majus, Crombie et al. 2002, Planta 214:406-413; from Oryza sativa, Nakai et al. 2007, J. Biochem 142:491-500). Apart from these another four α-xylosidases have been found in various species but their amino acid or nucleotide sequences have never been determined: Aspergillus flavus MO-5 α-xylosidase (Yoshikawa et al. 1993, Biosci. Biotechnol. Biochem. 57:1281-1285; Yoshikawa et al. 1993, Biosci. Biotechnol. Biochem. 58:121-125;); Pisum sativum α-xylosidase (O'Neill et al. 1989, J. Biol. Chem. 264:20430-20437); Bacillus α-xylosidase (Zong et al. 1989, Agric. Biol. Chem. 53:2129-2139); Aspergillus niger (Yoshikawa et al. 1994, Biosci. Biotechnol. Biochem. 58:1392-1398). All of these are less than 50% identical to the mature protein of SEQ ID NO: 2 herein.
A number of sequences have been published in the UNIPROT database (reference) that are indicated as Glycosyl hydrolases belonging to family 31 (EC=3.2.1.-;) or simply as “protein”. This includes also SEQ ID NOs: 2 and 3 as exemplified herein. The sequence identities of these sequences to SEQ ID NO: 2 range from 100% to 97.8%. Further related sequences (by sequence identity) are below 77.2% identity.
SEQ ID NO: 2 is thus published as UNIPROT:A7LZZ5 with the reference Fulton, L Clifton, S Fulton, B Xu, J Minx, P Pepin, K H Johnson, M Thiruvilangam, P Bhonagiri, V Nash, W E Mardis, E R Wilson, R K. Submitted (March-2007) to the EMBL/GenBank/DDBJ databases, and Sudarsanam, P Ley, R Guruge, J Turnbaugh, P J Mahowald, M Liep, D Gordon, J. Draft genome sequence of Bacteroides ovatus (ATCC 8483). Submitted (April-2007) to the EMBL/GenBank/DDBJ databases.
Sequences having between 99.2 and 97.8% identities to SEQ ID NO: 2 are disclosed as UNIPROT:I9STF6 (Bacteroides CL02T12C04), UNIPROT:D7J3T4 (Bacteroides sp. D22), UNIPROT:I9ADV6 (Bacteroides xylanisolvens CL03T12C04), UNIPROT:D4VSZ2 (Bacteroides ovatus SD CC 2a), UNIPROT:C3QER3 (Bacteroides sp 2_1_22), UNIPROT:D4WF36 (Bacteroides xylanisolvens SD CC 1b) and a few more
The present invention provides the use in animal feed of polypeptides having α-xylosidase activity and polynucleotides encoding the polypeptides. In comparison to other GH31 α-xylosidases acting on para nitro phenol α-D-xylose or on the X motif (Fry et al. 1993, Physiol. Plant. 89:1-3) on the non-reducing end of xylogluco oligosaccharides. The α-xylosidase in SEQ ID NO: 2 (GH31 α-xylosidase designated BACOVA 03422) is corn-xylan-specific and has a higher specific activity on corn xylan compared to CjXyl31.